Longevity extending compositions

Abstract

The present invention relates a composition for use in increasing the longevity and / or enhancing healthy aging of a subject, particularly a human subject, and methods thereof. The composition comprises at least one of: isoscabertopin; 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; eupafolin; isokuranetin, (E) - aldosecologanin, or any combination thereof, and increases expression of or activates a transcription factor selected from the group consisting of: nuclear factor (erythroid-derived 2)-like 2 (NRF2), Forkhead box O3 (FOXO3) and Grainy head 1 (GRHL1). Non-therapeutic or therapeutic uses and methods are disclosed herein. The extending longevity includes stimulating innate immunity, resistance against oxidative stress, inhibition of tumor cell growth, treating or preventing age-related diseases and disorders, which include: inflammation and inflammatory diseases, non-communicable diseases including: cardiovascular diseases, arthropathies, cancer, neurodegenerative diseases, diabetes, frailty, decline of physical fitness, decline of physical endurance or combinations thereof.

Description

LONGEVITY EXTENDING COMPOSITIONSField of the invention

[0001] The present invention relates to compositions for use in increasing the longevity of a subject, particularly a human subject, and methods thereof.Background of the invention

[0002] The dream of living forever permeates human culture. A longer life brings with it opportunities, not only for older people and their families, but for societies as a whole. It is predicted that by 2030, 1 in 6 people in the world will be aged 60 years or over. By 2050, the world's population of people aged 60 years and older will double to 2.1 billion. The number of persons aged 80 years or older is expected to triple between 2020 and 2050 to reach 426 million. (World Health Organization (WHO) - Ageing and health - 1 October 2022)

[0003] Yet, the opportunities associated with a longer life or increased longevity depends heavily on the concept of healthy aging. The WHO defines this concept as "the process of developing and maintaining the functional ability that enables wellbeing in older age."

[0004] Aging is an inevitable process, commonly defined as a gradual functional decline. Aging is characterized by a progressive loss of physiological integrity, accompanied by an increased risk of age-related diseases such as neurociegenerati ve disorders, arthropathies, cardiovascular diseases, type 2 diabetes and various cancers.

[0005] Aging is regarded as the outcome of a balance between cellular damage and repair (Haigis and Yanker, 2010). Lopez-Otin et al (2013) proposed nine Hallmarks of Aging: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication. Pathological dysfunctions of these nine processes are considered to accelerate aging in mammals, while the factors involved in regulation of these hallmarks may contribute to aging ( Lopez-Otin et al (2013); Martins et al (2016).

[0006] Aging is not only controlled by genetic factors, but also by exposure to stressors. A stressor can be defined as a chemical, biological, environmental, mental, or other internal or external stimuli which causes a threat to an individual. The ability to adequately respond to stressors is essential to maintain health, combat disease and maintain homeostasis which in turn leads to healthy longevity. Humans have evolved extensive networks of biological processes to maintain homeostasis. The so-called "stress response".

[0007] Cellular signalling pathways are at the core of the body's stress responses. These pathways can be triggered or upregulated by certain compositions and methods.

[0008] There have been various efforts to develop interventions to prevent or slow down the rate of biological aging, with the aim of extending life span in a healthy manner. Some efforts are more extreme than others. For example, following a Mediterranean diet, sleeping in a hyperbaric chamber, enjoying a daily glass of red wine, or editing a cells' genomic architecture via gene-therapy.

[0009] Consumers are seeking perceived healthy and natural solutions to healthy aging. The growing preference for food and beverage ingredients perceived as being "clean" is a global phenomenon. Natural ingredients ("nutraceuticals") extracted from plants, fruits and vegetables are favourable over pharmaceuticals.

[0010] Accordingly, there remains an unmet need for plant-derived compounds which can proactively counteract the aging process by targeting transcription factors involved in the stress response. There exists a need for novel, safe, and effective interventions for extending the life span of an individual in a healthy manner and / or increasing healthy years in your life (without necessarily extending years of lifespan). Improving the so- called "healthspan" of the individual. It would be preferable if the novel interventions were nutritional interventions, i.e., they involve nutritional compositions. It would be desirable if the compounds exhibited an effect in the treatment, reduction, and / or prevention of age-related diseases and disorders, such as: cardiovascular diseases, arthropathies, cancer, neurodegenerative diseases, diabetes and / or the like.Summary of the Invention

[0011] According to a first aspect of the invention, there is provided a composition for use in extending longevity and / or fostering healthy aging of a subject, wherein the composition comprises at least one of the following: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isosakuranetin, and e) (E) - aldosecologanin, or any combination thereof, and wherein the composition increases expression of or activates at least one of the transcription factors selected from the group consisting of: Forkhead box 03 (FOXO3) and Grainy head 1 (GRHL1) and optionally also, nuclear factor (erythroid-derived 2)-like 2 (NRF2).

[0012] In a second aspect of the invention, there is provided a longevity extending and / or healthy aging fostering composition, wherein the composition comprises an effective amount of a compound for increasing expression or activation of NRF2 transcription factor in a cell of a subject, wherein the compound is selected from the group consisting of isoscabertopin, 1,7-Bis (hydroxyphenyl)-4-hepten-3-one, eupafolin, and (E) - aldosecologanin, or combinations thereof.

[0013] In a third aspect of the invention, there is provided a longevity extending and / or healthy aging fostering composition comprising an effective amount of a compound for increasing expression or activation of FOXO3 transcription factor in a cell of a subject, wherein the compound is selected from the group consisting of isoscabertopin, eupafolin, and (E) - aldosecologanin, or combinations thereof.

[0014] In a fourth aspect of the invention, there is provided a longevity extending and / or healthy aging fostering composition comprising an effective amount of a compound for increasing expression or activation of GHRL transcription factor in a cell of a subject, wherein the compound is selected from the group consisting of isoscabertopin, 1,7-Bis(hydroxyphenyl)-4-hepten-3-one, isosakuranetin and (E) - aldosecologanin, or combinations thereof.

[0015] In a fifth aspect of the invention, there is provided the use of a composition in extending longevity and / or fostering healthy aging of a subject, wherein the composition comprises at least one of: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isosakuranetin, and e) (E) - aldosecologanin, or any combination thereof, and wherein the composition increases expression of or activates a transcription factor selected from the group consisting of: RHL1, FOXO3 and NRF2.

[0016] In a sixth aspect of the invention, there is provided the use of a composition in extending longevity and / or fostering healthy aging of a subject, wherein the composition comprises at least two of: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isosakuranetin, and e) (E) - aldosecologanin.

[0017] In some embodiments, the use is non-therapeutic. In some other embodiments, the use is therapeutic.

[0018] In some embodiments, the subject is a mammal, preferably a human.

[0019] In some embodiments the composition is a dietary supplement, nutritional composition, or nutritional product.

[0020] In some embodiments, the composition is a synthetic composition.Definitions

[0021] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one", but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or that the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. For example, but not by way of limitation, when the term "about" is utilized, the designated value may vary by plus or minus twelve percent, or eleven percent, or ten percent, or nine percent, or eight percent, or seven percent, or six percent, or five percent, or four percent, or three percent, or two percent, or one percent. The use of the term "at least one" will be understood to include one as well as any quantity more than one, including but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term to which it is attached; in addition, the quantities of 100 / 1000 are not to be considered limiting, as higher limits may also produce satisfactory results. In addition, the use of the term "at least one of X, Y, and Z" will be understood to include X alone, Y alone, and Z alone, as well as any combination of X, Y, and Z. The use of ordinal number terminology (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of differentiating between two or more items and is not meant to imply any sequence or order or importance to one item over another or any order of addition, for example. All ranges contained within this application are intended to include all numbers, whole or fractions, contained within said range.

[0022] The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth. The skilled artisan will understand that typically there is no limiton the number of items or terms in any combination, unless otherwise apparent from the context.

[0023] "Dietary Supplements" is a term used worldwide to represent a broad category of ingestible products that are distinguishable from conventional foods and drugs. The products are intended to supplement the diet.

[0024] As used herein, "effective amount" is preferably an amount that prevents a deficiency, treats a disease or medical condition in an individual or, more generally, reduces symptoms, manages progression of the diseases or provides a nutritional, physiological, or medical benefit to the individual. A treatment can be patient- or doctor- related. In addition, while the terms "individual" and "patient" are often used herein to refer to a human, the invention is not so limited. Accordingly, the terms "individual" and "patient" refer to any animal, mammal or human having or at risk for a medical condition that can benefit from the treatment.

[0025] "Healthy Aging" as used herein is a process of maintaining and improving physical and mental health, independence, and quality of life. It involves developing and maintaining functional abilities that allow for well-being especially in older age. These abilities include: meeting basic needs; learning, growing, and making decisions; being mobile; building and maintaining relationships; and, contributing to society. Healthy aging is best accomplished by focusing on four interconnected pillars: physical, social, emotional, and cognitive health. Some factors that can influence healthy aging include: Physical health which include staying active, reducing inflammation, absence of infections and / or (chronic) diseases, making and being able to have healthy food choices, getting enough and restful sleep, and proactively managing health care; Mental health: Managing social isolation, loneliness, stress, depression, and mood; Cognitive health: Healthy eating, staying active, and learning new skills.

[0026] "Life expectancy” means how long an individual is expected to live. This is affected by personal factors such as environment, diet, age, gender and genetics. The WHO reports that global average life expectancy is 73 years of age.

[0027] "Longevity" means how long an individual actually lives past the average life expectancy. Someone who has the capacity to survive past the average age of death is saidto have longevity. As used herein, "Longevity" is intended to include Healthy Aging, not solely longer life.

[0028] "Lifespan” means how long an individual could potentially live. Maximum lifespan is the oldest age a person could be reached by an individual under ideal conditions. This would require no disease, no accidents and perfect environmental conditions. Potential maximum lifespan is estimated to be between 115 and 125 years of age.

[0029] "Nutritional composition” means a substance or composition that satisfies at least a portion of a subject's nutrient requirements. "Nutritional composition(s)” may refer to Nutritional Products, liquids, powders, solutions, gels, pastes, solids, concentrates, suspensions, ready-to-use forms of enteral formulas, and / or oral formulas.

[0030] " Nutritional products" is understood to further include any number of optional additional ingredients, including conventional food additives, for example one or more, acidulants, additional thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifies, excipient, flavor agent, mineral, osmotic agents, a pharmaceutically acceptable carrier, preservatives, stabilizers, sugar, sweeteners, texturizers, and / or vitamin. The optional ingredients can be added in any suitable amount.

[0031] As used herein, "Subjects" include animals, which include, but is not limited to, mammals, which includes, but is not limited to: rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms subject, animal, or mammal, or their plurals are used, it is contemplated that it also applies to any animals that are capable of the effect exhibited or intended to be exhibited by the context of the passage.

[0032] The term "synthetic” when applied to a composition, a nutritional composition, or a mixture means a composition, nutritional composition, or mixture obtained by biological and / or chemical means, which can be chemically identical to the mixture naturally occurring in mammalian milks. A composition, nutritional composition, or mixture is said to be "synthetic” if at least one of its components is obtained by biological (e.g., enzymatic) and / or chemical means.

[0033] The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include, but are not limited to, individuals already having a particular condition / disease / infection as well as individuals who are at risk of acquiring a particular condition / disease / infection (e.g., those needing prophylactic / preventative measures). The term "treating" refers to administering an agent to a subject / patient for therapeutic and / or prophylactic / preventative purposes.

[0034] All percentages, parts, and ratios as used herein are detailed by weight of the total composition, unless otherwise specified. All amounts specified as administered “per day” may be delivered in a single unit dose, in a single serving, or in two or more doses or servings administered over the course of a 24-hour period.

[0035] All references to singular characteristics or limitations in the present disclosure shall include the corresponding plural characteristic or limitation, and vice versa, unless otherwise specified or clearly implied to the contrary, by the context in which the reference is made.

[0036] The compositions of the present disclosure can comprise, consist of, or consist essentially of any of the components described herein, as well as including any additional useful component.Detailed Description of the Invention

[0037] The present invention provides compositions for use in extending longevity and / or enhancing healthy aging of a subject, preferably a human subject.

[0038] The inventors of the present application have suprisingly found a number of plant- derived compounds that are able to activate one or more transcription factors known to be involved in a cell's stress response. The inventors have hypothesised that nutritional interventions that include one or more of these plant-derived compounds, will reduce cellular aging, and thereby extend an individual's longevity.

[0039] In order to determine suitable nutrtional intervention ingredients, the inventors conducted screening of Analyticon Discovery's MEGxp plant-derived compound libraryto identify those compounds with specificity for activation of the Forkhead box 03 (FOXO3) transcription factor, nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor and / or the Grainy head-like (GRH-1) transcription factor.

[0040] Transcription factors are molecules that control the activity of a gene by determining whether the gene's DNA (deoxyribonucleic acid) is transcribed into RNA (ribonucleic acid). This may result in increased or decreased protein synthesis, and subsequent altered cellular function.FOXO signaling

[0041] Forkhead box O (FOXO) proteins represent a subfamily of transcription factors conserved from Caenorhabditis elegans (C. elegans)' to mammals, that act as key regulators of longevity downstream of insulin and insulin-like growth factor. C. elegans possesses one FOXO representative, while in mammals there are four: FOXO1, FOXO3, FOXO4, and FOXO6.

[0042] FOXO proteins are the most important transcriptional effectors of the insulin-like growth factor 1 (IGF-1) signalling pathway (IIS), a pathway that is the most evolutionary conserved pathway of aging, across a great evolutionary distance from C. elegans to mice (Kenyon 1993). FOXO3 is a regulator of metabolic stress during aging. FOXO3 has been described as crucial factor for longevity, regulating major intracellular pathways such as gluconeogenesis, DNA repair, cell cycle arrest, stem cell homeostasis, immune system modulation, differentiation, cell death, apoptosis, redox balance, lipophagy, mitophagy, autophagy, and proteostasis.

[0043] Remarkably, the gene encoding the transcription factor for FOXO3 is currently one of very few individual genes with specific single nucleotide polymorphisms that are repeatedly linked to exceptional human longevity [Willcox, B.J., et al., FOXO3A genotype is strongly associated with human longevity. Proc Natl Acad Sci U S A, 2008. 105(37): p. 13987-92. Flachsbart, F., et al., Association of FOXO3A variation with human longevity confirmed in German centenarians. Proc Natl Acad Sci U S A, 2009. 106(8): p. 2700-5. Sanese, P., et al., FOXO3 on the Road to Longevity: Lessons From SNPs and Chromatin Hubs. Comput Struct Biotechnol J, 2019. 17: p. 737-745.]. Moreover, protein expression ofFOXO3 has been shown to decrease by 25% upon aging, while pharmacological activation thereof results in protection against age-related diseases and the promotion of healthy aging [10.1016 / j.arr.2022.101621 https: / / pubmed.ncbi.nlm.nih.gov / 35421606 / ].Nrf2 signaling

[0044] Reactive oxygen species (ROS) are critical contributors to aging. ROS modulate cellular function by modifying proteins, lipids, carbohydrates and nucleic acids, altering their stability and function. If not eliminated, the accumulation of ROS causes cellular oxidative stress.

[0045] Numerous endogenous cellular anti-oxidant systems have evolved to ensure that ROS are properly reduced. This includes the NRF2-KEAPI pathway.

[0046] Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a member of a family of basic leucine transcription factors that binds to Antioxidant Response Elements (AREs) in the promotor regions of genes including those involved in redox regulation, DNA repair and prevention of apoptosis (Hayes, 2014).

[0047] Arguably one of the most prominent evolutionarily conserved transcription factors repeatedly linked to aging and age-related disease progression, in particular tumorigenesis, NRF2 is commonly referred to as "the" master regulator of redox homeostasis and oxidative stress responses [Hayes, J.D. and A.T. Dinkova-Kostova, The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem Sci, 2014. 39(4): p. 199-218. Menegon, S., A. Columbano, and S. Giordano, The Dual Roles of NRF2 in Cancer. Trends Mol Med, 2016. 22(7): p. 578-593. Vomund, S., et al., Nrf2, the Master Regulator of Anti-Oxidative Responses. Int J Mol Sci, 2017. 18(12).].

[0048] While conclusive evidence for the involvement of NRF2 in human aging and associated pathologies is still missing, NRF2 expression was found to be altered in autopsy brain tissues from human patients of Alzheimer's and Parkinson's disease [Ramsey, C.P., et al., Expression of Nrf2 in neurodegenerative diseases. J Neuropathol Exp Neurol, 2007. 66(1): p. 75-85.]. NRF2 expression has also been shown to be suppressed in a cellularmodel of Hutchinson-Gilford progeria syndrome, a rare disorder that causes premature aging in children, with reactivation of NRF2 partially rescuing several of the phenotypical defects [Kubben, N., et al., Repression of the Antioxidant NRF2 Pathway in Premature Aging. Cell, 2016. 165(6): p. 1361-1374.].GRHL signaling

[0049] Grainy head-like transcription factors are evoluhonarily conserved from fungi to humans. GRH-1 in C. elegans and GRHL1-3 in mammals have previously been described as crucial players during embryonic development and cancer [He, J.X., et al., Grainyhead- like 2 as a double-edged sword in development and cancer. American Journal of Translational Research, 2020. 12(2): p. 310-331.]. Recently, a link has been identified between GRH- 1 / GRHL1 signaling and aging [Grigolon, G., et al., Grainyhead 1 acts as a drug-inducible conserved transcriptional regulator linked to insulin signaling and lifespan. Nat Commun, 2022. 13(1): p. 107. Rozanov, L., et al., Redox-mediated regulation of aging and healfhspan by an evoluhonarily conserved transcription factor HLH-2 / Tcf3 / E2A. Redox Biol, 2020. 32: p. 101448.]. Both overexpression and pharmacological activation of GRHL1 leads to lifespan extension in C. elegans. Furthermore, GRH-1 / GRHL1 promotes improved pathogen defense in C. elegans and counteracts cellular senescence in murine primary fibroblasts [Grigolon, G., et al., Grainyhead 1 acts as a drug-inducible conserved transcriptional regulator linked to insulin signaling and lifespan. Nat Commun, 2022. 13(1): p. 107.]

[0050] Based on the above-identified knowledge of the role of the FOXO3, NRF2 and GRL1-3 transcription factors in the aging process, the inventors hypothesise that plant- derived compounds which are able to activate and / or increase the expression of one or more of these transcription factors will be useful in extending the longevity of a subject, particularly a human subject. Further, it is hypothesised that a composition comprising a combination of plant-derived compounds, each of which target a different and / or the same transcription factor, will result in a synergistic effect in extending the longevity of a subject.

[0051] To determine suitable plant-derived compounds, the inventors initially screened 2215 compounds from Analyticon Discovery's MEGxp compound library, in a luciferasereporter line assay to identify compounds that activated at least one of: FOXO3, NRF2 and GRL1-3. The different plant-derived compounds identified as potential nutritional interventions, for each transcription factor, are detailed below.

[0052] Table 1 - Overview of the compounds that activate one or more of FOXO3, NRF2 and GRL1-3 transcription factors.Isoscabertopin

[0053] Isoscabertopin (CAS No. 439923-16-17) is a sesquiterpene lactone that can be isolated from Elephantopus scaber (Prickly Leaved Elephant's Foot). E. scaber Einn family Asteraceae, is a tropical species of flowering plant in the Asteraceae family and is a genus of perennial herb widely spread in Asia, Tropical Africa, India and Australia. E. scaber has been used in traditional medicine as an astringent agent, cardiac tonic, antidiuretic, antiviral and antibacterial agent. Sesquiterpene lactones are abundant in all E. scaber plant parts. Isoscabertopin has been identified as a potent anticancer compound (Xu, 2006).

[0054] Using a Human Embryonic Kidney (HEK) luciferase reporter cell assay, the inventors have identified that isoscabertopin activates the transcription factors NRF2, FOXO3 and GHRL2.

[0055] To the best of the inventors' knowledge, isoscabertopin has never previously been identified as a plant derived nutritional intervention for extending longevity in a subject, particularly a human subject.1,7-Bis (hydroxyphenyl)-4-hepten-3-one

[0056] 1,7-Bis (hydroxyphenyl)-4-hepten-3-one (CAS No. 59673-65-0) is a simple aromatic organic compound. This phytochemical can be extracted from Alnus Ruba (Red alder) a deciduous broadleaf tree native to western North America, and also from Betula platyphylline (Asian white birch), one of the most widely distributed temperate deciduous tree species in East Asia. 1,7-Bis (hydroxyphenyl)-4-hepten-3-one has been identified as inducing apoptosis by suppressing autophagy flux and activating the p38 pathway in lung cancer cells (Jung et al, 2020)

[0057] Using a HEK luciferase reporter cell assay, the inventors have identified that 1,7- Bis (hydroxyphenyl)-4-hepten-3-one activates the transcription factors GRHL and NRF2.

[0058] To the best of the inventors' knowledge, 1,7-Bis (hydroxyphenyl)-4-hepten-3-one has never previously been identified as a plant derived nutritional intervention for extending longevity in a subject, particularly a human subject.Eupafolin

[0059] Eupafolin (6-methoxy-5,7 / 3',4'-tetrahydroxyflavone) (CAS no. 520-11-6), also known as nepetin, is the major active flavonoid isolated from common sage Salvia officinalis'). Eupafolin has been shown to exert anti-inflammatory and anti-proliferative effects in cell lines (Chen et al, 2016).

[0060] Using a HEK luciferase reporter cell assay, the inventors have identified that eupafolin the transcription factors FOXO3 and NRF2.

[0061] To the best of the inventors' knowledge, eupafolin has never previously been identified as a plant derived nutritional intervention for extending longevity in a subject, particularly a human subject.Isosakuranetin

[0062] Isosakuranetin (CAS No. 26207-61-4) is a racemate of two enantiomers, the S(-) and R(-) enantiomers, and is also referred to as 4'-methyloxy-5,7-dihydroxyflavanone. It is a flavanone substituted by hydroxy groups at positions 5 and 7 and a methoxy group at position 4' (the 2S stereoisomer). Isosakuranetin is closely related to 4-methyl-naringenin.

[0063] Citrus fruit and juices represent one of the main sources of compounds with a high potential for health promoting properties. Among these compounds, flavanones (such as hesperetin, naringenin, eriodictyol, isosakuranetin, and their respective glycosides), which occur in quantities ranging from ~180 to 740 mg / L (depending on the Citrus species and cultivar) are responsible for many biological activities. Evidence demonstrates that these compounds may support and enhance the body's defenses against oxidative stress and help the organism in the prevention of cardiovascular diseases, atherosclerosis, and cancer (Barreca, D., et al. (2017)).

[0064] Using a HEK luciferase reporter cell assay, the inventors have identified that isosakuranetin activates the transcription factors GHRL2.

[0065] To the best of the inventors' knowledge, Isosakuranehn has never previously been identified as a plant derived nutritional intervention for extending longevity in a subject, particularly a human subject.(E) - Aldosecologanin

[0066] (E) - Aldosecologanin (CAS No. 471271-55-3) is a natural iridoid glycoside found in the stems and leaves of Lonicera japonica (honeysuckle).

[0067] Using a HEK luciferase reporter cell assay, the inventors have identified that (E) - Aldosecologanin activates the transcription factors NRF2, FOXO3 and GHRL2.

[0068] To the best of the inventors' knowledge, (E) - Aldosecologanin has never previously been identified as a plant derived nutritional intervention for extending longevity in a subject, particularly a human subject.EXPERIMENTAL PROCEDURE1. Primary Compounds Screening for GRHL, FOXO and / or NRF2 transcription factor activation1.1 METHODOLOGY

[0069] For all screening purposes in tissue culture, in-house generated HEKluciferase reporter cells were used, containing synthetic promoter elements with several repeats of mammalian GRHL1-3, FOXO3 or NRF2 binding sequences. The synthetic promoters control the expression of a firefly luciferase reporter gene. Increased expression of this reporter gene, assayed using a luciferase substrate, corresponds to activation of transcription factor (GRHL, FOXO, NRF2) signalling. In brief, the screening was performed as follows:

[0070] Day 1: Seeding of reporter cell lines in 96-well plates a. 'Luciferase' reporter cell lines1) Seeding of FOXO reporter cell line at 12,500 cells I well in a total volume of 100 pl DMSO / well.2) Seeding of GRHL reporter cell line at 50,000 cells / well in a total volume of 100 pl DMSO / well.3) Seeding of NRF2 reporter cell line at 25,000 cells I well in a total volume of 100 pl DMSO / well.4) Duplicates were seeded in plates for protein assessment 'Protein plates' for validation assays via SRB (Sulforhodamine B (SRB) assay.

[0071] Day 2: Stimulation of reporter cell lines with compounds from Analyticon Discovery's MEGxp compound library

[0072] Stimulation of 'Luciferase' (and 'Protein' plates) reporter cell lines with compounds from Analyticon Discovery's MEGxp compound library was undertaken by adding 25 pl of an appropriate compound pre-dilution per well. For the primary compound screening, the final concentration per well was 10 pM of each compound.

[0073] Positive controls:GRHL activator: scriptaid (5 pM per well)FOXO activator: resveratrol (5 pM per well).The positive controls used are very effective and highly sensitive. Therefore 5 pM was determined to be the optimal dose for the positive controls vs a higher concentration of 10 pM for the compounds to assess the efficacy and sensitivity of the subject compounds.

[0074] Day 3: Assessment of activation using ONEGlo ™ Luciferase Assay System

[0075] Readout was performed after 24 h incuba tion with the compounds, using the ONEGlo™ Luciferase Assay System (Promega Corporation, #E6130) reagent and the CLARIOstar microplate reader (BMG LABTECH) according to the manufacturers' instructions.

[0076] Day 3: Protein assessment - Sulforhodamine B (SRB) assay

[0077] The SRB assay is based on the ability of the protein dye SRB to bind electrostatically and pH-dependent to protein basic amino acid residues of trichloroacetic acid-fixed cells. Under mild basic conditions the dye can be extracted from cells and quantified by photometric or fluorescent determination.

[0078] Sulforhodamine B (SRB) assay with 'protein plate' for validation assays only: To precipitate the proteins in the plates, the cell culture medium was removed and the cells incubated with 150 pl ice-cold TCA (10% ) for 45-60 min at 4°C. After that, the 96 well plates were washed 5 times with tab water and set aside for drying. Meanwhile, the sulforhodamine B (SRB) (Santa Cruz; #sc-253615A) solution with 0.4 g in 100 ml of 1% acetic acid solution was prepared. Dried wells were then incubated with 70 pl SRB solution for 15 min at RT. To remove residual SRB, the plate was washed 5 to 6 times with 100 pl of 1 % acetic acid per well. The plate was dried again for 4 h at 37°C or overnight at RT. Finally, 100 pl of unbuffered Tris was added to each well and the absorbance was measured at 510 nm with a CLARIOstar microplate reader (BMG LABTECH) after an incubation of 15 min at 4°C. 3) The raw data were then normalized to DMSO solvent control (for initial screening) or to DMSO solvent control and protein levels (for validation assays).RESULTS

[0079] Primary screening of 2220 compound library for GRHL and FOXO activation

[0080] The raw data were normalized to DMSO. The top 160 activators were identified by applying a threshold of 2.5-fold GRHL and 1.5-fold FOXO activation.

[0081] As Nrf2 is a well-described transcription factor involved in aging, we included a Nrf2-activation assay with the top 80 GRHL and / or FOXO activating compounds. Nrf2- activating compounds were then further tested in Nrf2 Luciferase Reporter cells lacking Nrf2 (Nrf2 KO), to evaluate the specificity of luciferase signal (Fig. 4). We defined a > 50% luciferase signal in Nrf2 KO compared to Nrf2 WT well as Nrf2-unspecific signal and excluded these structures from the list of Nrf2-activating compounds.

[0082] Protein Validation assays of 160 primary hits for GHRL and FOXO activation

[0083] The validation assays of the top 160 compounds were repeated on GRHL and FOXO reporter cells in triplicates and were normalized to protein. The top 80 activators were identified by applying a threshold of 8-fold for GRHL only activators and 3-fold for FOXO only activators. Compounds that activated both transcription factors needed a 3- fold activation for both transcription factors to be considered as "hits". Initial screening was done with a single replicate and without normalization to protein.

[0084] Secondary screening of 160 primary hits for GRHL, and FOXO

[0085] Cell assays were repeated in order to identify the top 80 activators of GRHL, and / or FOXO. The top activators were identified by applying a threshold of 8-fold activation for GRHL, a 3-fold activation for FOXO activation over the DMSO control. Compounds that activated GRHL, and FOXO transcription factors needed a 3-fold activation for each transcription factors to be considered as a "hit". GRHL, and FOXO reporters have very different sensitivities. For example, the positive controls have different activation strengths. The GRHL reporter is the most sensitive, which is why we chose a higher threshold of 8-fold compared to the FOXO at 3-fold.

[0086] Determination of top 80 GRHL and / or FOXO activators for NRF2 activation

[0087] As Nrf2 is a well-described transcription factor involved in aging, we included an Nrf2-activation assay with the top 80 GRHL and / or FOXO activating compounds. Nrf2 activation was identified by applying a threshold of 3-fold activation.

[0088] In order to validate the transcription factor activity reported above in wild-type cells, and to exclude the possibility that the activation of the luciferase reporter occurred in the absence of the actual transcription factor, further cell assays were conducted with the top 80 compounds for (a) the activation of GRHL in wild-type (WT) and GRHL- deficient (knock-out; KO) reporter cells, and (b) the activation of NRF2 in wild-type (WT) and NRF2-deficient (knock-out; KO) reporter cells,

[0089] To the best of our knowledge, there is no functional and viable FOXO knockout cell line published to date, presumably due to crucial cellular functions of FOXO. Hence the assays could not be repeated for FOXO activating compounds in a FOXO-deficient background. Of note, Abeam does provide a FOXO3 KO cell line (ab260857), however, this product has no peer reviews and is not subject of any published article yet. Additionally, worms and flies are believed to act as viable FOXO knockouts, but no validated and peer reviewed cell lines as of this application.

[0090] The luciferase signal of compound-treated WT cells and KO cells were compared. A > 50% luciferase signal in KO cells compared to WT cells was defined as an unspecific signal. These compounds were excluded from the list of GRHL- and / or NRF2 activating compounds.

[0091] Details of the activation of FOXO, GRHL and NRF2 for the 5 exemplary compounds according to the invention are provided in Table 2. The data relates to the fold-change compared to the DMSO control.

[0092] Table 2: Transcription factor activation (fold-change compared to DMSO) of the 5 exemplary compounds according to the invention. It is contemplated that FOXO maybe toxic at higher (50 pM) concentrations.C. elegans assaysMethods

[0093] C. elegans culture - C. elegans was cultured at 20°C on nematode growth media (NGM) seeded with E. coli OP50. Strains that were obtained from Caenorhabditis Genetics Center (University of Minnesota, USA) included Bristol N2 (wild type), EU31 skn-1 (zul35), CF1038 daf-16 (mu86), VC2072 grh-1 (gk960).

[0094] Bacteria preparation - Alive bacteria were prepared for C. elegans maintenance as previously described [Rozanov, L., et al., Redox-mediated regulation of aging and healthspan by an evolutionarily conserved transcription factor HLH-2 / Tcf3 / E2A. Redox Biol, 2020. 32: p. 101448.]. E. coli OP50 bacteria (CGC) were streaked out on DYT agar plates and single colonies picked from such plates were cultured overnight at 37 °C and constant shaking in Erlenmeyer flasks containing liquid DYT medium. Bacterial overnight cultures were concentrated by centrifugation for 30 min at 3200 x g and 4 °C. The prepared bacteria were spotted on NGM agar plates and allowed to grow for 16-24 h prior to use.

[0095] Heat inactivated (HIT) OP50 were prepared for C. elegans compound lifespans as previously described [Grigolon, G., et al., Grainyhead 1 acts as a drug-inducible conserved transcriptional regulator linked to insulin signaling and lifespan. Nat Commun, 2022. 13(1) : p. 107.] . In short, the overnight OP50 culture was pelleted by centrifugation as above, all DYT media removed, and bacteria resuspended in S-buffer supplemented with 1 M MgSO4 and 5 mg / ml Cholesterol to have a 10-fold concentrated culture. Afterwards, the bacterial suspension was placed in a 65 °C water bath for 45 min. HIT OP50 were spotted on NGM agar plates on the day of use and dried for 30 min before adding the worms.

[0096] For the lifespan assay with toxic bacteria M. nematophilum (CGC, #CBX102) was grown for 48 h in liquid LB medium at 37 °C and diluted 10-fold with OP50 before being spotted on NGM agar plates.

[0097] C. elegans lifespan and survival assays - The compound or DMSO (solvent control) was added into autoclaved liquid hot NGM (50°C) prior to plate pouring. Plates were allowed to solidify for three days at room temperature before use. Adult nematodes were allowed to lay eggs for four to nine hours and the resulting eggs incubated for 64 h at 20 °C on NGM agar plates inoculated with OP50 to obtain a synchronized population of young adult nematodes. Synchronized L4 stage worms were transferred to compoundcontaining NGM plates inoculated with HIT-OP50 if not otherwise indicated. For a typical lifespan assay, 100-120 young adult nematodes per condition were manually transferred to NGM agar plates (35-40 nematodes per 55 mm petri dish). For the first 10-12 days, nematodes were transferred daily and afterwards every 2-3 days. Nematodes showing no reaction to gentle stimulation were scored as dead. Nematodes that crawled off the plates, displayed internal hatching, or a protruding vulva were censored.

[0098] In case of the toxic bacteria assay, alive bacteria were used. Additionally, the food source was switched from OP-50 to M. nematophilum from day seven of adulthood onwards. In case of the paraquat stress assay, the nematodes were additionally treated with 5 mM paraquat (Sigma Aldrich; #56177) from day three of adulthood onwards. Dead worms were counted in 10-14 h steps.

[0099] Bending Assay in C. elegans - Synchronized L4 nematodes were transferred to compound-containing NGM plates. On day 5 of adulthood 10-20 worms were transferred into a drop of S-buffer and their bending movement was recorded for 30 s with a Leicasystem (Leica M165FC microscope with Leica camera DFC 3000G). At least 20 nematodes per condition were analyzed via automatized analysis using the ImageJ plugin "wrMTrck"[Hahm, J.H., et al., 2015. 6: p. 8919.].

[0100] Quantification of mtDNA copy number - Total DNA for mtDNA quantification was extracted from grinded nematodes on Day 5 of compound treatment by standard proteinase K and phenol-chloroform extraction. mtDNA / nDNA levels were quantified in at least three biological replicates using SYBR Green select master mix (Applied Bio systems) fluorescence on a 96-well format in CFX96 real time system (Biorad). mtDNA / nDNA ratios were calculated by 2x2 dCT method[Venegas, V. and M.C. Halberg, 2012.]. Used primers are based on previous publications [Venegas, V. and M.C. Halberg, 2012; Gonzalez-Hunt, C.P., et al., 2016; Hoogewijs, D., et al., 2008]

[0101] Cancer cell proliferation assay - MCF-7 cells (#300273, CLS Cell Lines Service GmbH) were cultured with DMEM (Sigma Aldrich; #D6429) and 4.5 g / 1 glucose, phenol red, 10% FBS, and 1% penicillin / streptomycin with pH 7.4 and were incubated at 37°C, 5% CO2, and 95% relative humidity. Cells were seeded into a 96 well plate and allowed to attach for 24 h. Treatment was performed by addition of the compound to the cell culture media. Ice-cold TCA (10%) was used for protein precipitation at timepoint 0 and 96 h after treatment start. Proteins were stained with 0.4% Sulforhodamine B (SRB) (SantaCruz; #sc- 253615A) in 1% acetic acid, washed, and dissolved in Tris. Absorbance was measured at 510 nm with a CLARIOstar microplate reader (BMG LABTECH).Results

[0102] A comprehensive literature search was done and selection of the top 9 compounds was made by evaluating effectivity as FOXO, GRHL and / or NRF2 activators in combination with novelty, by and excluding all compounds already described as FOXO, GRHL and / or NRF2 activators and all compounds that are already major focus of the aging process / age-related diseases.

[0103] C. elegans lifespan (dose-response) - To further investigate potential health- beneficial effects of selected compounds, lifespan assays were performed in C. elegans by supplementation of the respective compound into the NGM agar plates. Thereby, a broad range of concentrations was applied using 10-fold dilutions between 10 nM and 10 pM.

[0104] As a result, we elucidated five out of nine structures as longevity factors, consisting of three "main selection" and two "backup" compounds (Fig. 1, for statistics see Table 3). All these phytochemicals showed a mean lifespan extension between 8-12 % with varying results in maximal lifespan extension in either one or several tested concentrations. In contrast, the remaining four compounds did not have any effect on C. elegans lifespan (Fig- 2).Key for Figures 1 to 11Figure 1: Compounds that extend lifespan in C. elegans in either one or several concentrations between 10 nM and 10 pM. These compounds include A) Isoscabertopin, B) l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, C) Eupafolin, D) Isosakuranehn, and E) Aldosecologanin. Statistics: log-rank test.Figure 2: Compounds that do not affect lifespan in C. elegans in either one or several concentrations between 10 nM and 10 pM. These compounds include A) NA / Inventa (NP-015955), B) Garcinoic Acid, C) 5, 6, 7-Trihydroxy-4' -methoxyflavone, and D) 7-(4- Hydroxy-3-methoxyphenyl)-l-phenyl-4-hepten-3-one. Statistics: log-rank test.Figure 3: C. elegans bending frequency upon supplementation of compounds in their lifespan-extending concentration(s). A-C) C. elegans bending frequency is increased upon supplementation with A) 10 nM isoscabertopin, B) 1 pM of l,7-Bis(4-hydroxyphenyl)-4- hepten-3-one, and C) 1 and 10 pM Eupafolin. D-E) In contrast, 10 pM of either D) isosakuranehn or E) aldosecologanin results in a decrease in bending frequency. Statistics: One-way ANOVA with Dunnett Posthoc test (panel A-C) and two-sided unpaired student's t-test (panel D and E).Figure 4: C. elegans mitochondrial DNA (mtDNA) content upon supplementation of compounds in their lifespan-extending concentration. While l,7-Bis(4-hydroxyphenyl)- 4-hepten-3-one shows a significant increase in mitochondrial mass on day 5 of treatment, isoscabertopin, eupafolin, and aldosecologanin do not have such an effect. Statistics: Oneway ANOVA with Dunnett Posthoc test.Figure 5: Transcription factor dependency of isoscabertopin in C. elegans lifespan assays. Isoscabertopin (10 nM) extends lifespan in A) WT (=N2) C. elegans but not in B)GRHL (grh-1 ), C) FOXO (= DAF-16), or D) NRF2 (= skn-l)-deficient nematodes. Statistics: log-rank test.Figure 6: Transcription factor dependency of l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one in C. elegans lifespan assays. l / 7-Bis(4-hydroxyphenyl)-4-hepten-3-one (1 pM) extends lifespan in A) WT (=N2) C. elegans and C) FOXO-deficient (= DAF-16) nematodes, but not in B) GRHL (grh-1), or D) NRF2 (= skn-l)-deficient nematodes. Statistics: log-rank test.Figure 7: Transcription factor dependency of eupafolin in C. elegans lifespan assays. Eupafolin (10 ApM) extends lifespan in A) WT (=N2) C. elegans, B) GRHL-deficient (= grh- 1) and D) NRF2 (= skn-l)-deficient but not C) FOXO-deficient (= DAF-16) nematodes. Statistics: log-rank test.Figure 8: Transcription factor dependency of isosakuranetin in C. elegans lifespan assays. Isosakuranetin (10 pM) extends lifespan in A) WT (=N2) C. elegans, C) FOXO (= DAF-16), and D) NRF2-(= skn-l)-deficient nematodes but not in B) GRHL (grh-1) - deficient C. elegans. Statistics: log-rank test.Figure 9: Toxic bacteria (M. nematophilum) immunity assay in C. elegans upon treatment with top 5 compounds in their lifespan-extending concentration. These compounds include A) isoscabertopin (10 nM), B) l,7-Bis(4-hydroxyphenyl)-4-hepten-3- one (1 pM), C) eupafolin (10 pM),and D) isosakuranetin (10 pM). Statistics: log-rank test.Figure 10: Paraquat stress assay in C. elegans upon treatment with top 5 compounds in their lifespan-extending concentration. These compounds include A) Isoscabertopin (10 nM), B) l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one (1 pM), C) Eupafolin (10 pM), D) Isosakuranetin (10 pM), and E) Aldosecologanin (100 nM). Statistics: log-rank test.Figure 11: MCF-7 tumor cell proliferation upon treatment with top 5 compounds after 96 h of treatment. While l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, isoscabertopin, eupafolin, and isosakuranetin inhibit proliferation in at least one tested concentration, aldosecologanin did not have such an effect. Statistics: Brown-Forsythe and Welch ANOVA with Dunett T3 multiple comparisons Posthoc test.

[0105] Table 3 Log-rank statistics to compound lifespans in wild-type and mutant C. elegans.

[0106] C. elegans exercise performance and metabolism - Improved exercise performance has been linked to increased health and the interference with anti-aging pathways [Memme, J.M., et al., 2021.; Ristow, M., et al., 2009.].

[0107] Consequently, we supplemented our five lifespan-extending compounds to C. elegans and analysed the nematode's movement speed by quantification of their bending frequency on day 5 of treatment (Fig. 3).

[0108] We observed that three out of five compounds - namely isoscabertopin, 1,7-Bis(4- hy roxyphenyl)-4-hepten-3-one, and eupafolin - led to a significant increase in bending frequency in at least one of the tested life-extending concentrations. In contrast, isosakuranehn and aldosecologanin nematodes bent slower than control worms.

[0109] Increased exercise performance is often linked with an increased number of energy-producing mitochondria [Merry, T.L. and M. Ristow, Nuclear factor erythroid- derived 2-like 2 (NFE2L2, Nrf2) mediates exercise-induced mitochondrial biogenesis and antioxidant response in mice. J Physiol, 2016. 15(594): p. 5195-207, Memme, J.M., et al., Exercise and mitochondrial health. J Physiol, 2021. 599(3): p. 803-817.]. Consequently, we quantified the mitochondrial DNA (mtDNA) copy number in C. elegans lysates upon five days of compound treatment as parameter for overall mitochondrial mass (Fig. 4).

[0110] We observed that l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one significantly increased the mtDNA copy number by 42 percent, while all other compounds did not show similar effects.

[0111] Transcription factor-dependency in C. elegans lifespan assays - We performed lifespan analyses with nematodes lacking either of these transcription factors (Fig. 5-9; for statistics see Table 4).

[0112] Table 4: Log-rank statistics to compound survival assays in wild-type C. elegans.

[0113] Isoscabertopin is a potent triple activator of FOXO (5-fold), GRHL (19-fold), and NRF2 (53-fold) in HEK293 luciferase reporter cell. In accordance with this, lifespan extension was abolished when applying isoscabertopin (10 nM) to GRHL-deficient (= grh- 1) (Fig. 5B), FOXO deficient (= DAF-16) (Fig. 5C), or NRF2-deficient (= skn-1) (Fig. 5D) nematodes compared to long-lived wild-type (= N2) (Fig. 5A) C. elegans.

[0114] l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one is a potent activator of GRHL (83-fold), and NRF2 (65-fold) but not FOXO (<l-fold) in HEK293 luciferase reporter cell lines (see Report B). In accordance with this, lifespan extension was abolished when applying 1,7- Bis(4-hydroxyphenyl)-4-hepten-3-one (1 pM) to GRHL-deficient (= grh-1) (Fig. 6B), or NRF2-deficient (= skn-1) (Fig. 6D) nematodes compared to long-lived wild-type (= N2) (Fig. 6 A) C. elegans and FOXO deficient (= DAF-16) (Fig. 6C) nematodes.

[0115] Eupafolin is a potent activator of FOXO (6-fold), and NRF2 (7-fold) but not GRHL (2-fold) in HEK293 luciferase reporter cell lines (see Report B). In accordance with this, lifespan extension was abolished when applying eupafolin (10 pM) to FOXO-deficient (= DAF-16) (Fig. 7C) nematodes compared to long-lived wild-type (= N2) (Fig. 7A) C. elegans and GRHL-deficient (= grh-1) (Fig. 7B) nematodes. However, lifespan is still extended in NRF2-deficient (= skn-1) nematodes (Fig. 7D), suggesting that the relative mild activation seen in cell assays is not sufficient to regulate lifespan in C. elegans.

[0116] Isosakuranetin is a potent activator of GRHL (66-fold) but not NRF2 (1.4-fold) nor FOXO (1.7-fold) in HEK293 luciferase reporter cell lines. In accordance with this, lifespan extension was abolished when applying isosakuranetin (10 pM) to GRHL-deficient (= grh- 1) (Fig. 8B) nematodes compared to long-lived wild-type (= N2) (Fig. 8A) C. elegans, as well as FOXO-deficient (= DAF-16) (Fig. 8C) and NRF2-deficient (= skn-1) (Fig. 8D) nematodes.

[0117] Immunity towards toxic bacteria - We performed lifespan experiments with A4. nematophilum as bacterial food source starting from day 7 of adulthood onwards. A4. nematophilum is harmful to nematodes due to immune suppression in the nematode's gut, thereby significantly impairing the host's immune system and lifespan [ O'Rourke, D„ et al., 2006.].

[0118] Three out of four tested compounds promoted survival of nematodes exposed to M. nematophilum significantly (Fig. 9). While isoscabertopin, l,7-Bis(4-hydroxyphenyl)-4- hepten-3-one and eupafolin rescued the harmful impact of these toxic bacteria, isosakuranehn did not alter C. elegans lifespan and thus might not improve protection against pathogen infection. Note that due to technical issues of the exclusive supplier of aldosecologanin, it was not included in this lifespan assay.

[0119] Oxidative stress resistance towards paraquat exposure - We performed survival experiments upon paraquat treatment (5 mM) starting from day three of adulthood onwards. Paraquat is a strong reactive oxygen (ROS) generator and thus causes oxidative stress leading to cellular damage and a profound decrease in health- and lifespan.

[0120] As a result, three out of the five tested compounds promoted survival in nematodes exposed to paraquat significantly (Fig. 10, Table 5). While isoscabertopin, eupafolin, and isosakuranetin increased survival under severe oxidative stress, 1,7-Bis(4- hydroxyphenyl)-4-hepten-3-one and aldosecologanin did not have such an effect and thus might not improve oxidative stress resistance in C. elegans.

[0121] Table 5: Lead structures and their evaluation in health assays.

[0122] Inhibition of cancer cell growth in MCF-7 cells - We performed a proliferation assay with the MCF-7 breast tumor cell line. The purpose of this experiment was toidentify compounds with the ability to interfere with tumor signaling pathways, thereby building the fundament for further characterization as anti-tumor agents.

[0123] Four out of the five tested compounds showed significant impairment of tumor proliferation after 96 hours of treatment (Fig. 11). While isoscabertopin and 1,7-Bis(4- hydroxyphenyl)-4-hepten-3-one have a half maximal inhibitory concentration (IC50) of <10 pM, IC50 values of eupafolin and isosakuranetin exceed 10 pM and 50 pM, respectively. Aldosecologanin did not interfere with cell proliferation at any tested concentration.

[0124] In summary, to elucidate novel health-inducing small molecules as activators of anti-aging modulators such as FOXO3, GRHL1, and NRF2, we have screened a compound library including 2,400 phytochemicals from Traditional Chinese Medicine (TCM) in cellbased luciferase reporter systems and identified five hit structures namely eupafolin (activator of FOXO and NRF2), l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one (activator of GRHL and NRF2), isoscabertopin (activator of FOXO, GRHL and NRF2), isosakuranetin (activator of GRHL), and aldosecologanin (activator of FOXO, GRHL and NRF2). Interestingly, these structures prolonged lifespan in the nematode C. elegans dependent on the presence of the transcription factors they were previously demonstrated to trigger in human cells. Further characterization revealed that three of these structures, namely l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, isoscabertopin, and eupafolin have a profound health-promoting influence on physical activity, protection from pathogen infection, and resistance against oxidative stress in C. elegans as well as inhibition of tumor cell growth in MCF-7 cells. In conclusion, the herein described structures have profound antiaging properties in cells and C. elegans and further preclinical evaluation might establish them as effective dietary supplements against negative alignments of aging.

[0125] Thus, in accordance with the present disclosure, there have been provided compositions, as well as methods of producing and using same, which fully satisfy the objectives and advantages set forth hereinabove. Although the present disclosure has been described in conjunction with the specific drawings, experimentation, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit andbroad scope of the present disclosure. It should be appreciated that the present invention is not limited to the specific embodiments described above, but includes variations, modifications and equivalent embodiments defined by the following claims. The following claims are to be understood to be presented for the purposes of illustration only and do not in any way limit the scope of the inventive concept(s) described or otherwise contemplated herein.References1. Artal-Sanz, M., L. de Jong, and N. Tavernarakis, Caenorhabditis elegans: a versatile platform for drug discovery. Biotechnol J, 2006. 1(12): p. 1405-18.2. Barreca, D., et al. (2017). "Flavanones: Citrus phytochemical with health-promoting properties." Biofactors 43(4): 495-5063. Chen C-C, Lin M-W, Laing C-J, Wang S-H (2016). The anti-inflammatory effects and mechanisms of eupafolin in lipopolysaccharide induced inflammatory responses in RAW264.7 macrophages. PLoS One: ll(7):e0158662.4. Gonzalez-Hunt, C.P., et al., PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage. Curr Protoc Toxicol, 2016. 67: p. 2011 1-201125.5. Grigolon, G., et al., Grainyhead 1 acts as a drug-inducible conserved transcriptional regulator linked to insulin signaling and lifespan. Nat Commun, 2022. 13(1): p. 107.6. Hahm, J.H., et al., C. elegans maximum velocity correlates with healthspan and is maintained in worms with an insulin receptor mutation. Nat Commun, 2015. 6: p. 8919.7. Haigis, M. C., and Yankner, B. A. (2010). The aging stress response. Mol. Cell. 40, 333-344.8. Hayes JD and Dinkova-Kostova AT, The Nrf2 regulatory network provides an interface between redox and intermediary metabolism. Trends Biochem Sci, 2014 39(4): p. 199-218.9. Hoogewijs, D., et al., Selection and validation of a set of reliable reference genes for quantitative sod gene expression analysis in C. elegans. BMC Mol Biol, 2008. 9: p. 9.10. Jung, H. J., et al. (2020). "l,7-Bis(4-hydroxyphenyl)-4-hepten-3-one from Betula platyphylla induces apoptosis by suppressing autophagy flux and activating the p38 pathway in lung cancer cells." Phytother Res 34(1): 126-138Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R (1993) A C. elegans mutant that lives twice as long as wild type. Nature 366, 461 Lee, J.E., Diet Before and After Breast Cancer. Adv Exp Med Biol, 2021. 1187: p. 545- 566. Lopez-Otin, C., Blasco, M. A., Partridge, L., Serrano, M., and Kroemer, G. (2013). The hallmarks of aging. Cell 153, 1194-1217. Martins, R., Lithgow, G. J., and Link, W. (2016). Long live FOXO: unravelling the role of FOXO proteins in aging and longevity. Aging Cell. 15, 196-207. Memme, J.M., et al., Exercise and mitochondrial health. J Physiol, 2021. 599(3): p. 803- 817. Merry, T.L. and M. Ristow, Nuclear factor erythroid-derived 2-like 2 (NFE2L2, Nrf2) mediates exercise-induced mitochondrial biogenesis and antioxidant response in mice. J Physiol, 2016. 15(594): p. 5195-207. O'Rourke, D., et al., Genomic clusters, putative pathogen recognition molecules, and antimicrobial genes are induced by infection ofC. elegans with M. nematophilum. Genome Res, 2006. 16(8): p. 1005-16. Ristow, M., et al., Antioxidants prevent health-promoting effects of physical exercise in humans. Proc Nat Acad Sci, 2009. 106(21): p. 8665-70. Rozanov, L., et al., Redox-mediated regulation of aging and healthspan by an evolutionarily conserved transcription factor HLH-2 / Tcf3 / E2A. Redox Biol, 2020. 32: p. 101448. Tanner, C.M., et al., Rotenone, Paraquat and Parkinson's Disease. Environ Health Perspect, 2011. 119(6): p. 866-72. Venegas, V. and M.C. Halberg, Measurement of mitochondrial DNA copy number. Methods Mol Biol, 2012. 837: p. 327-35. WHO factsheet: Aging and Health. October 2022. Xu, G., et al. (2006). Antitumor activities of the four sesquiterpene lactones from Elephantopus scaber L. Exp Oncol 28(2): 106-109

Claims

CLAIMS1. A composition for use in extending longevity of a subject, wherein the composition comprises at least one of the following: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isosakuranetin, and e) (E) - aldosecologanin, or any combination thereof, and wherein the composition increases expression of or activates at least one transcription factor selected from the group consisting of: nuclear factor (erythroid-derived 2)-like 2 (NRF2), Forkhead box 03 (FOXO3), and Grainy head 1 (GRHL1).

2. The composition for use according to claim 1, wherein the composition comprises at least two of: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isokuranetin, and e) (E) - aldosecologanin.

3. The composition for use according to claim 1 or 2, wherein the use is a non- therapeutic use.

4. The composition for use according to claim 1, 2, or 3 wherein the subject is a mammal, preferably a human.

5. A longevity extending composition comprising an effective amount of a compound for increasing expression or activation of NRF2 transcription factor in a cell of a subject, wherein the compound is selected from the group consisting of isoscabertopin, 1,7-Bis (hydroxyphenyl)-4-hepten-3-one, eupafolin, and (E) - aldosecologanin, or combinations thereof.

6. A longevity extending composition comprising an effective amount of a compound for increasing expression or activation of FOXO3 transcription factor in a cell of a subject, wherein the compound is selected from the group consisting of isoscabertopin, eupafolin, and (E) - aldosecologanin, or combinations thereof.

7. A longevity extending composition comprising an effective amount of a compound for increasing expression or activation of GHRL transcription factor in a cell of a subject, wherein the compound is selected from the group consisting of isoscabertopin, 1,7-Bis (hydroxyphenyl)-4-hepten-3-one, isokuranetin and (E) - aldosecologanin, or combinations thereof.

8. The composition according to any one of claims 1 to 7, wherein the composition is a dietary supplement, or a nutritional composition.

9. The use of a composition for extending longevity of a subject, wherein the composition comprises at least one of: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isokuranetin, and e) (E) - aldosecologanin, or any combination thereof, and wherein the composition increases expression of or activates at least one transcription factor selected from the group consisting of: nuclear factor (erythroid-derived 2)-like 2 (NRF2), Forkhead box 03 (FOXO3), and Grainy head 1 (GRHL1).

10. The use of claim 9, wherein the composition comprises at least two of: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isokuranetin, and e) (E) - aldosecologanin.

11. The use of claim 9, wherein the use is non-therapeutic or therapeutic.

12. The composition for use of claim 1 and / or the use of claim 9, wherein the extending longevity is treating or preventing age-related diseases and disorders.

13. osition for use according to claim 12 and / or the use of claim 12, wherein the d diseases and disorders consist of: cardiovascular diseases, arthropathies, urodegenerative diseases, diabetes, or combinations thereof.

14. The use of claim 12, wherein the extending longevity is stimulating innate immunity, resistance against oxidative stress, inhibition of tumor cell growth, or combinations thereof.

15. The use of claim 12, wherein the treating or preventing age-related diseases and disorders is stimulating innate immunity, resistance against oxidative stress, inhibition of tumor cell growth, or combinations thereof.

16. The use according to claims 9 - 15 wherein extending longevity comprises extending how long an individual lives.

17. The use according to claims 9 - 15 wherein extending longevity comprises extending healthy aging.

18. The use according to claim 17 wherein extending healthy aging comprises maintaining and / or improving physical health, mental health, independence, quality of life, or combinations thereof.

19. The use according to claim 18 wherein physical health comprises staying active, reducing inflammation, absence of infections, absence of chronic diseases, absence of acute diseases, getting enough sleep, getting restful sleep, or combinations thereof.

20. The use according to claim 18 wherein mental health comprises managing social isolation, loneliness, stress, depression, mood, or combinations thereof.

21. A method for extending longevity of a subject comprising administering a composition to the subject, wherein the composition comprises at least one of the following: a) isoscabertopin; b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isosakuranetin, and e) (E) - aldosecologanin, or any combination thereof, and wherein the composition increases expression of or activates at least one transcription factor selected from the group consisting of: nuclear factor (erythroid-derived 2)-like 2 (NRF2), Forkhead box 03 (FOXO3), and Grainy head 1 (GRHE1).

22. The method of claim 21, wherein the composition comprises at least two of: a) isoscabertopin;b) 1,7-Bis (hydroxyphenyl)-4-hepten-3-one; c) eupafolin; d) isokuranetin, and e) (E) - aldosecologanin.

23. The method of claim 21 or 22, wherein the extending longevity is treating or preventing age-related diseases and disorders.

24. The method of claim 23, wherein the age-related diseases and disorders consist of: cardiovascular diseases, arthropathies, cancer, neurodegenerative diseases, diabetes, or combinations thereof.

25. The method of claim 23 or 24, wherein the extending longevity is stimulating innate immunity, resistance against oxidative stress, inhibition of tumor cell growth, or combinations thereof.

26. The method of any one of claims 23 to 25, wherein the treating or preventing age-related diseases and disorders is stimulating innate immunity, resistance against oxidative stress, inhibition of tumor cell growth, or combinations thereof.

27. The method of any one of claims 21 to 26, wherein extending longevity comprises extending how long an individual lives.

28. The method of any one of claims 21 to 27, wherein extending longevity comprises extending healthy aging.

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